Platelet-derived growth factor compositions and methods of use thereof

ABSTRACT

A method for promoting growth of bone, periodontium, ligament, or cartilage in a mammal by applying to the bone, periodontium, ligament, or cartilage a composition comprising platelet-derived growth factor at a concentration in the range of about 0.1 mg/mL to about 1.0 mg/mL in a pharmaceutically acceptable liquid carrier and a pharmaceutically-acceptable solid carrier.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/159,533,filed Jun. 23, 2005, which issued as U.S. Pat. No. 7,473,678, which is acontinuation-in-part of application Ser. No. 10/965,319, filed Oct. 14,2004, now abandoned, both herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the healing of bone and connective tissues.

BACKGROUND OF THE INVENTION

Growth factors are proteins that bind to receptors on a cell surface,with the primary result of activating cellular proliferation and/ordifferentiation. Many growth factors are quite versatile, stimulatingcellular division in numerous different cell types; while others arespecific to a particular cell-type. Examples of growth factors includeplatelet-derived growth factor (PDGF), insulin-like growth factors IGF-Iand II), transforming growth factor beta (TGF-β), epidermal growthfactor (EGF), and fibroblast growth factor (FGF). PDGF is a cationic,heat stable protein found in a variety of cell types, including thegranules of circulating platelets, vascular smooth muscle cells,endothelial cells, macrophage, and keratinocytes, and is known tostimulate in vitro protein synthesis and collagen production byfibroblasts. It is also known to act as an in vitro mitogen andchemotactic agent for fibroblasts, smooth muscle cells, osteoblasts, andglial cells.

Recombinant human PDGF-BB (rhPDGF-BB) has been shown to stimulate woundhealing and bone regeneration in both animals and humans. It is approvedin both the United States and Europe for human use in topicalapplications to accelerate healing of chronic diabetic foot sores.Recombinant hPDGF-BB has also been shown to be effective either singlyor in combination with other growth factors for improving periodontalregeneration, i.e., regrowth of bone, cementum, and ligament aroundteeth (see, e.g., U.S. Pat. No. 5,124,316, incorporated herein byreference).

SUMMARY OF THE INVENTION

We have now demonstrated that a low dose of rhPDGF (˜0.1 to 1.0 mg/mL)promotes repair of bone, periodontium, ligament, and cartilage. A lowamount of rhPDGF can be adsorbed to β-TCP, which can be implanted at thesite of repair, such that the rhPDGF is released in vivo. Addition ofrhPDGF to β-TCP has been shown to enhance osteoblast cell attachment andproliferation compared to untreated β-TCP.

In a first aspect, the invention features a method for promoting bone,periodontium, ligament, or cartilage growth in a mammal, e.g., a human,by administering an implant material containing platelet-derived growthfactor (PDGF) at a concentration of less than about 1.0 mg/ml, such thatthe implant material promotes growth of the bone, periodontium,ligament, or cartilage. In an embodiment, the PDGF is administered in anamount of less than or equal to 0.3 mg/ml. In another embodiment, thePDGF is administered in an amount in the range of about 0.1 to about 1.0mg/ml. In several embodiments, the PDGF is administered in an amount ofbetween about 0.2 to about 0.75 mg/ml, about 0.25 to about 0.6 mg/ml,and about 0.25 to about 0.5 mg/ml. In an embodiment, the PDGF isadministered in an amount of about 0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml,preferably 0.3 mg/mL. In another embodiment, the PDGF is eitherpartially or substantially purified. In yet a further embodiment, thePDGF is isolated or purified from other contaminants. In a furtherembodiment, the PDGF is released from the implant material uponadministration at an average rate of 0.3 mg/day. In another embodiment,the PDGF is released from the implant material upon administration at anaverage rate of 300 μg/day. In still further embodiments, the PDGF isreleased from the implant material at an average rate of less than 100μg/day, less than 50 μg/day, less than 10 μg/day, or less than 1 μg/day.Preferably, the PDGF is delivered over a few days, e.g., 1, 2, 5, 10,15, 20, or 25 days, or up to 28 days or more.

A second aspect of the invention features a method for promoting bone,periodontium, ligament, or cartilage growth in a mammal, e.g., a human,by administering an implant material containing an amount ofplatelet-derived growth factor (PDGF) of less than about 1.0 mg/ml and apharmaceutically acceptable carrier such that the implant materialpromotes the growth of the bone, periodontium, ligament, or cartilage,and allowing the bone, periodontium, ligament, or cartilage to grow.Preferably, the PDGF is equal to or less than about 0.3 mg/ml. In anembodiment, the PDGF is administered in a range of about 0.1 to 1.0mg/ml. In other embodiments, the amount of PDGF is about 0.1 mg/ml, 0.3mg/ml, or 1.0 mg/ml, preferably 0.3 mg/mL. In another embodiment, thePDGF is either partially or substantially purified. In yet a furtherembodiment, the PDGF is isolated or purified from other contaminants.Prior to administering the implant material to the mammal, the methodcan additionally include the step of producing a surgical flap of skinto expose the bone, periodontium, ligament, or cartilage, and followingthe administration step, replacing the flap. In yet another embodiment,after producing the surgical flap, but prior to administering theimplant material to the bone, periodontium, ligament, or cartilage, themethod can additionally include the step of planing the bone orperiodontium to remove organic matter from the bone or periodontium. Inyet another embodiment, the method promotes the growth of damaged ordiseased bone, periodontium, ligament, or cartilage. In yet anotherembodiment, the method promotes the growth of bone in locations wherenew bone formation is required as a result of surgical interventions,such as, e.g., tooth extraction, ridge augmentation, esthetic grafting,and sinus lift.

A third aspect of the invention features an implant material forpromoting the growth of bone, periodontium, ligament, or cartilage in amammal, e.g., a human. The implant material includes a pharmaceuticallyacceptable carrier (e.g., a biocompatible binder, a bone substitutingagent, a liquid, or a gel) and platelet-derived growth factor (PDGF),which is present at a concentration of less than about 1.0 mg/mL.Preferably, the PDGF is present in the implant material at aconcentration equal to or less than about 0.3 mg/ml. In an embodiment,the PDGF is administered in a range of about 0.1 to 1.0 mg/ml. In otherembodiments, the amount of PDGF is about 0.1 mg/ml, 0.3 mg/ml, or 1.0mg/ml, preferably 0.3 mg/mL. In an embodiment, the pharmaceuticallyacceptable carrier of the implant material includes a scaffold or matrixconsisting of a biocompatible binder (e.g., carboxymethylcellulose) or abone substituting agent (β-TCP) that is capable of absorbing a solutionthat includes PDGF (e.g., a solution containing PDGF at a concentrationin the range of about 0.1 mg/mL to about 1.0 mg/mL). In anotherembodiment, the pharmaceutically acceptable carrier is capable ofabsorbing an amount of the PDGF solution that is equal to at least about25% of its own weight. In other embodiments, the pharmaceuticallyacceptable carrier is capable of absorbing an amount of the PDGFsolution that is equal to at least about 50%, 75%, 100%, 200%, 250%, or300% or its own weight. In an embodiment, the PDGF is absorbed by thepharmaceutically acceptable carrier of the implant material by soakingthe pharmaceutically acceptable carrier in a solution containing PDGF.Preferably, the PDGF is present in the solution at a concentration ofless than about 1.0 mg/mL. In another embodiment, the PDGF is present inthe solution at a concentration equal to or less than about 0.3 mg/ml.In another embodiment, the PDGF is present in the solution at aconcentration in the range of about 0.1 to 1.0 mg/ml. In yet otherembodiments, the PDGF is present in the solution in an amount of about0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml, preferably 0.3 mg/mL. In anotherembodiment, the PDGF is either partially or substantially purified. Inyet a further embodiment, the PDGF is isolated or purified from othercontaminants.

A fourth aspect of the invention features a method for preparing animplant material for promoting growth of bone, periodontium, ligament,or cartilage in a mammal, e.g., a human. The method includes the step ofcombining partially purified or purified platelet-derived growth factor(PDGF) in an amount of less than about 1.0 mg/mL with a pharmaceuticallyacceptable carrier substance. Preferably, the PDGF is combined with apharmaceutically acceptable carrier substance at a concentration equalto or less than about 0.3 mg/ml. In an embodiment, the PDGF is combinedwith a pharmaceutically acceptable carrier substance in an amount in therange of about 0.1 to 1.0 mg/ml. In other embodiments, PDGF is mixed inthe amount of 0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml. In another embodiment,PDGF is mixed in the amount of 0.3 mg/ml. In yet another embodiment, thePDGF is absorbed by the pharmaceutically acceptable carrier to producethe implant material.

A fifth aspect of the invention features a vial having platelet-derivedgrowth factor (PDGF) at a concentration in the range of about 0.1 mg/mLto about 1.0 mg/mL in a pharmaceutically acceptable liquid. In anembodiment of this aspect of the invention, the liquid is sterile sodiumacetate buffer. In another embodiment, the vial contains PDGF at aconcentration of about 0.3 mg/mL. In yet another preferred embodiment,the PDGF is PDGF-BB. In yet other embodiments, the PDGF is stable in thesodium acetate buffer for at least about 12 months, preferably at leastabout 18 months, more preferably at least about 24 months, and mostpreferably at least about 36 months when stored at a temperature in therange of about 2° C. to 80° C.

A sixth aspect of the invention features an implant material thatincludes a porous calcium phosphate having adsorbed therein a liquidcontaining platelet-derived growth factor (PDGF) at a concentration inthe range of about 0.1 mg/mL to about 1.0 mg/mL. In several embodiments,the concentration of PDGF is about 0.3 mg/mL, the calcium phosphate isselected from tricalcium phosphate, hydroxyapatite, poorly crystallinehydroxyapatite, amorphous calcium phosphate, calcium metaphosphate,dicalcium phosphate dihydrate, heptacalcium phosphate, calciumpyrophosphate dihydrate, calcium pyrophosphate, and octacalciumphosphate, and the PDGF is provided in a sterile liquid, for example,sodium acetate buffer.

A seventh aspect of the invention features a method of preparing animplant material by saturating a calcium phosphate material in a sterileliquid that includes platelet-derived growth factor (PDGF) at aconcentration in the range of about 0.1 mg/mL to about 1.0 mg/mL. Inseveral embodiments, the concentration of PDGF is about 0.3 mg/mL, andthe calcium phosphate is selected from tricalcium phosphate,hydroxyapatite, poorly crystalline hydroxyapatite, amorphous calciumphosphate, calcium metaphosphate, dicalcium phosphate dihydrate,heptacalcium phosphate, calcium pyrophosphate dihydrate, calciumpyrophosphate, and octacalcium phosphate.

In an embodiment of all aspects of the invention, PDGF includes PDGFhomo- and heterodimers, for example, PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC,and PDGF-DD, and combinations and derivatives thereof.

In an embodiment of all aspects of the invention, the pharmaceuticallyacceptable carrier substance of the implant material is or additionallyincludes one or more of the following: a biocompatible binder (e.g., anatural or synthetic polymer), a bone substituting agent, a liquid, anda gel. In another preferred embodiment, the implant material includesPDGF present in a pharmaceutically acceptable liquid carrier which isadsorbed by a pharmaceutically acceptable solid carrier.

In another embodiment of all aspects of the invention, the implantmaterial is prepared by combining isolated, partially purified,substantially purified, or purified PDGF in an amount in the range of0.1 to 1.0 mg/ml, more preferably 0.1 mg/ml, 0.3 mg/ml, or 1.0 mg/ml,most preferably 0.3 mg/ml, or even less than 0.1 mg/ml, with apharmaceutically acceptable carrier substance, e.g., a biocompatiblebinder, such as a natural or synthetic polymer (e.g., collagen,polyglycolic acid, and polylactic acid), a bone substituting agent(e.g., a calcium phosphate (e.g., tricalcium phosphate orhydroxyapatite), calcium sulfate, or demineralized bone (e.g.,demineralized freeze-dried cortical or cancellous bone), or acommercially available gel or liquid (i.e., a viscous or inert gel orliquid).

In several embodiments, the carrier substance of the implant materialis, or additionally includes, one or more biocompatible binders. Abiocompatible binder is an agent that produces or promotes cohesionbetween the combined substances. Non-limiting examples of suitablebiocompatible binders include polymers selected from polysaccharides,nucleic acids, carbohydrates, proteins, polypeptides, poly(α-hydroxyacids), poly(lactones), poly(amino acids), poly(anhydrides),poly(orthoesters), poly(anhydride-co-imides), poly(orthocarbonates),poly(α-hydroxy alkanoates), poly(dioxanones), poly(phosphoesters),polylactic acid, poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA),polyglycolide (PGA), poly(lactide-co-glycolide (PLGA),poly(L-lactide-co-D, L-lactide), poly(D,L-lactide-co-trimethylenecarbonate), polyglycolic acid, polyhydroxybutyrate (PHB),poly(ε-caprolactone), poly(δ-valerolactone), poly(γ-butyrolactone),poly(caprolactone), polyacrylic acid, polycarboxylic acid,poly(allylamine hydrochloride), poly(diallyldimethylammonium chloride),poly(ethyleneimine), polypropylene fumarate, polyvinyl alcohol,polyvinylpyrrolidone, polyethylene, polymethylmethacrylate, carbonfibers, poly(ethylene glycol), poly(ethylene oxide), poly(vinylalcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, poly(ethyleneterephthalate)polyamide, and copolymers and mixtures thereof. Additionalbinders include alginic acid, arabic gum, guar gum, xantham gum,gelatin, chitin, chitosan, chitosan acetate, chitosan lactate,chondroitin sulfate, N,O-carboxymethyl chitosan, a dextran (e.g.,α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or sodium dextransulfate), fibrin glue, glycerol, hyaluronic acid, sodium hyaluronate, acellulose (e.g., methylcellulose, carboxy methylcellulose, hydroxypropylmethylcellulose, or hydroxyethyl cellulose), a glucosamine, aproteoglycan, a starch (e.g., hydroxyethyl starch or starch soluble),lactic acid, a pluronic, sodium glycerophosphate, collagen, glycogen, akeratin, silk, and derivatives and mixtures thereof. In someembodiments, the biocompatible binder is water-soluble. A water-solublebinder dissolves from the implant material shortly after itsimplantation in vivo, thereby introducing macroporosity into the implantmaterial. This macroporosity increases the osteoconductivity of theimplant material by enhancing the access and, consequently, theremodeling activity of the osteoclasts and osteoblasts at the implantsite.

The biocompatible binder may be added to the implant material in varyingamounts and at a variety of stages during the preparation of thecomposition. Those of skill in the art will be able to determine theamount of binder and the method of inclusion required for a givenapplication.

In an embodiment, the carrier substance is or includes a liquid selectedfrom water, a buffer, and a cell culture medium. The liquid may be usedin any pH range, but most often will be used in the range of pH 5.0 topH 8.0. In an embodiment, the pH will be compatible with the prolongedstability and efficacy of the PDGF present in the implant material, orwith the prolonged stability and efficacy of another desiredbiologically active agent. In most embodiments, the pH of the liquidwill be in the range of pH 5.5 to pH 7.4. Suitable buffers include, butare not limited to, carbonates, phosphates (e.g., phosphate bufferedsaline), and organic buffers such as Tris, HEPES, and MOPS. Most often,the buffer will be selected for its biocompatibility with the hosttissues and its compatibility with the biologically active agent. Formost applications in which nucleic acids, peptides, or antibiotics areincluded in the implant material, a simple phosphate buffered salinewill suffice.

In another embodiment of all aspects of the invention, the carriersubstance of the implant material is, or additionally includes, one ormore bone substituting agents. A bone substituting agent is one that canbe used to permanently or temporarily replace bone. Followingimplantation, the bone substituting agent can be retained by the body orit can be resorbed by the body and replaced with bone. Exemplary bonesubstituting agent include, e.g., a calcium phosphate (e.g., tricalciumphosphate (e.g., β-TCP), hydroxyapatite, poorly crystallinehydroxyapatite, amorphous calcium phosphate, calcium metaphosphate,dicalcium phosphate dihydrate, heptacalcium phosphate, calciumpyrophosphate dihydrate, calcium pyrophosphate, and octacalciumphosphate), calcium sulfate, or demineralized bone (e.g., demineralizedfreeze-dried cortical or cancellous bone)). In an embodiment, thecarrier substance is bioresorbable. In another embodiment, the bonesubstituting agent is provided as a matrix of micron- or submicron-sizedparticles, e.g., nano-sized particles. The particles can be in the rangeof about 100 μm to about 5000 μm in size, more preferably in the rangeof about 200 μm to about 3000 μm, and most preferably in the range ofabout 250 μm to about 2000 μm, or the particles can be in the range ofabout 1 nm to about 1000 nm, preferably less than about 500 nm, and morepreferably less than about 250 nm. In another embodiment, the bonesubstituting agent has a porous composition. Porosity of the compositionis a desirable characteristic as it facilitates cell migration andinfiltration into the composition so that the cells can secreteextracellular bone matrix. It also provides access for vascularization.Porosity also provides a high surface area for enhanced resorption andrelease of active substances, as well as increased cell-matrixinteraction. Preferably, the composition has a porosity of greater than40%, more preferably greater than 65%, and most preferably greater than90%. The composition can be provided in a shape suitable forimplantation (e.g., a sphere, a cylinder, or a block) or it can be sizedand shaped prior to use. In a preferred embodiment, the bonesubstituting agent is a calcium phosphate (e.g., β-TCP).

The bone substituting agent can also be provided as a flowable, moldablepaste or putty. Preferably, the bone substituting agent is a calciumphosphate paste that self-hardens to form a hardened calcium phosphateprior to or after implantation in vivo. The calcium phosphate componentof the invention may be any biocompatible calcium phosphate materialknown in the art. The calcium phosphate material may be produced by anyone of a variety of methods and using any suitable starting components.For example, the calcium phosphate material may include amorphous,apatitic calcium phosphate. Calcium phosphate material may be producedby solid-state acid-base reaction of crystalline calcium phosphatereactants to form crystalline hydroxyapatite solids. Other methods ofmaking calcium phosphate materials are known in the art, some of whichare described below.

The calcium phosphate material can be poorly crystalline apatitic (PCA)calcium phosphate or hydroxyapatite (HA). PCA material is described inapplication U.S. Pat. Nos. 5,650,176; 5,783,217; 6,027,742; 6,214,368;6,287,341; 6,331,312; and 6,541,037, all of which are incorporatedherein by reference. HA is described, for example, in U.S. Pat. Nos. Re.33,221 and Re. 33,161. These patents teach preparation of calciumphosphate remineralization compositions and of a finely crystalline,non-ceramic, gradually resorbable hydroxyapatite carrier material basedon the same calcium phosphate composition. A similar calcium phosphatesystem, which consists of tetracalcium phosphate (TTCP) and monocalciumphosphate (MCP) or its monohydrate form (MCPM), is described in U.S.Pat. Nos. 5,053,212 and 5,129,905. This calcium phosphate material isproduced by solid-state acid-base reaction of crystalline calciumphosphate reactants to form crystalline hydroxyapatite solids.

Crystalline HA materials (commonly referred to as dahllite) may beprepared such that they are flowable, moldable, and capable of hardeningin situ (see U.S. Pat. No. 5,962,028). These HA materials (commonlyreferred to as carbonated hydroxyapatite) can be formed by combining thereactants with a non-aqueous liquid to provide a substantially uniformmixture, shaping the mixture as appropriate, and allowing the mixture toharden in the presence of water (e.g., before or after implantation).During hardening, the mixture crystallizes into a solid and essentiallymonolithic apatitic structure.

The reactants will generally consist of a phosphate source, e.g.,phosphoric acid or phosphate salts, substantially free of water, analkali earth metal, particularly calcium, source, optionally crystallinenuclei, particularly hydroxyapatite or calcium phosphate crystals,calcium carbonate, and a physiologically acceptable lubricant, such asany of the non-aqueous liquids described herein. The dry ingredients maybe pre-prepared as a mixture and subsequently combined with thenon-aqueous liquid ingredients under conditions where substantiallyuniform mixing occurs.

The calcium phosphate material is characterized by its biologicalresorbability, biocompatibility, and its minimal crystallinity. Itscrystalline character is substantially the same as natural bone.Preferably, the calcium phosphate material hardens in less than fivehours, and substantially hardens in about one to five hours, underphysiological conditions. Preferably, the material is substantiallyhardened within about 10-30 minutes. The hardening rate underphysiological conditions, may be varied according to the therapeuticneed by modifying a few simple parameters as described in U.S. Pat. No.6,027,742, which is incorporated herein by reference.

In an embodiment, the resulting bioresorbable calcium phosphate materialwill be “calcium deficient,” with a calcium to phosphate molar ratio ofless than about 1.6 as compared to the ideal stoichiometric value ofapproximately 1.67 for hydroxyapatite.

Desirable calcium phosphates are capable of hardening in a moistenvironment, at or around body temperature in less than 5 hours andpreferably within 10-30 minutes. Desirable materials are those that,when implanted as a 1-5 g pellet, are at least 80% resorbed within oneyear. Preferably, the material can be fully resorbed.

In several embodiments of all aspects of the invention, the implantmaterial additionally may include one or more biologically activeagents. Biologically active agents that can be incorporated into theimplant materials of the invention include, without limitation, organicmolecules, inorganic materials, proteins, peptides, nucleic acids (e.g.,genes, gene fragments, gene regulatory sequences, and antisensemolecules), nucleoproteins, polysaccharides, glycoproteins, andlipoproteins. Classes of biologically active compounds that can beincorporated into the implant materials of the invention include,without limitation, anti-cancer agents, antibiotics, analgesics,anti-inflammatory agents, immunosuppressants, enzyme inhibitors,antihistamines, anti-convulsants, hormones, muscle relaxants,anti-spasmodics, ophthalmic agents, prostaglandins, anti-depressants,anti-psychotic substances, trophic factors, osteoinductive proteins,growth factors, and vaccines.

Anti-cancer agents include alkylating agents, platinum agents,antimetabolites, topoisomerase inhibitors, antitumor antibiotics,antimitotic agents, aromatase inhibitors, thymidylate synthaseinhibitors, DNA antagonists, farnesyltransferase inhibitors, pumpinhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNF alphaagonists/antagonists, endothelin A receptor antagonists, retinoic acidreceptor agonists, immuno-modulators, hormonal and antihormonal agents,photodynamic agents, and tyrosine kinase inhibitors.

Any of the biologically active agents listed in Table 1 can be used.

TABLE 1 Alkylating agents cyclophosphamide lomustine busulfanprocarbazine ifosfamide altretamine melphalan estramustine phosphatehexamethylmelamine mechlorethamine thiotepa streptozocin chlorambuciltemozolomide dacarbazine semustine carmustine Platinum agents cisplatincarboplatinum oxaliplatin ZD-0473 (AnorMED) spiroplatinum, lobaplatin(Aeterna) carboxyphthalatoplatinum, satraplatin (Johnson Matthey)tetraplatin BBR-3464 (Hoffmann-La Roche) ormiplatin SM-11355 (Sumitomo)iproplatin AP-5280 (Access) Antimetabolites azacytidine tomudexgemcitabine trimetrexate capecitabine deoxycoformycin 5-fluorouracilfludarabine floxuridine pentostatin 2-chlorodeoxyadenosine raltitrexed6-mercaptopurine hydroxyurea 6-thioguanine decitabine (SuperGen)cytarabin clofarabine (Bioenvision) 2-fluorodeoxy cytidine irofulven(MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) idatrexateethynylcytidine (Taiho) Topoisomerase amsacrine rubitecan (SuperGen)inhibitors epirubicin exatecan mesylate (Daiichi) etoposide quinamed(ChemGenex) teniposide or mitoxantrone gimatecan (Sigma-Tau) irinotecan(CPT-11) diflomotecan (Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecinTAS-103 (Taiho) topotecan elsamitrucin (Spectrum) dexrazoxanet(TopoTarget) J-107088 (Merck & Co) pixantrone (Novuspharma) BNP-1350(BioNumerik) rebeccamycin analogue (Exelixis) CKD-602 (Chong Kun Dang)BBR-3576 (Novuspharma) KW-2170 (Kyowa Hakko) Antitumor dactinomycin(actinomycin D) amonafide antibiotics doxorubicin (adriamycin) azonafidedeoxyrubicin anthrapyrazole valrubicin oxantrazole daunorubicin(daunomycin) losoxantrone epirubicin bleomycin sulfate (blenoxane)therarubicin bleomycinic acid idarubicin bleomycin A rubidazonebleomycin B plicamycinp mitomycin C porfiromycin MEN-10755 (Menarini)cyanomorpholinodoxorubicin GPX-100 (Gem Pharmaceuticals) mitoxantrone(novantrone) Antimitotic paclitaxel SB 408075 (GlaxoSmithKline) agentsdocetaxel E7010 (Abbott) colchicine PG-TXL (Cell Therapeutics)vinblastine IDN 5109 (Bayer) vincristine A 105972 (Abbott) vinorelbine A204197 (Abbott) vindesine LU 223651 (BASF) dolastatin 10 (NCI) D 24851(ASTAMedica) rhizoxin (Fujisawa) ER-86526 (Eisai) mivobulin(Warner-Lambert) combretastatin A4 (BMS) cemadotin (BASF)isohomohalichondrin-B (PharmaMar) RPR 109881A (Aventis) ZD 6126(AstraZeneca) TXD 258 (Aventis) PEG-paclitaxel (Enzon) epothilone B(Novartis) AZ10992 (Asahi) T 900607 (Tularik) IDN-5109 (Indena) T 138067(Tularik) AVLB (Prescient NeuroPharma) cryptophycin 52 (Eli Lilly)azaepothilone B (BMS) vinflunine (Fabre) BNP-7787 (BioNumerik)auristatin PE (Teikoku Hormone) CA-4 prodrug (OXiGENE) BMS 247550 (BMS)dolastatin-10 (NIH) BMS 184476 (BMS) CA-4 (OXiGENE) BMS 188797 (BMS)taxoprexin (Protarga) Aromatase aminoglutethimide exemestane inhibitorsletrozole atamestane (BioMedicines) anastrazole YM-511 (Yamanouchi)formestane Thymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias)synthase inhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagoniststrabectedin (PharmaMar) mafosfamide (Baxter International) glufosfamide(Baxter International) apaziquone (Spectrum Pharmaceuticals) albumin +32P (Isotope Solutions) O6 benzyl guanine (Paligent) thymectacin(NewBiotics) edotreotide (Novartis) Farnesyltransferase arglabin(NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors lonafarnib(Schering-Plough) perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer)Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (EliLilly) tariquidar (Xenova) biricodar dicitrate (Vertex) MS-209 (ScheringAG) Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan)acetyltransferase SAHA (Aton Pharma) depsipeptide (Fujisawa) inhibitorsMS-275 (Schering AG) Metalloproteinase Neovastat (Aeterna Laboratories)CMT-3 (CollaGenex) inhibitors marimastat (British Biotech) BMS-275291(Celltech) Ribonucleoside gallium maltolate (Titan) tezacitabine(Aventis) reductase inhibitors triapine (Vion) didox (Molecules forHealth) TNF alpha virulizin (Lorus Therapeutics) revimid (Celgene)agonists/antagonists CDC-394 (Celgene) entanercept (Immunex Corp.)infliximab (Centocor, Inc.) adalimumab (Abbott Laboratories) EndothelinA atrasentan (Abbott) YM-598 (Yamanouchi) receptor antagonist ZD-4054(AstraZeneca) Retinoic acid fenretinide (Johnson & Johnson) alitretinoin(Ligand) receptor agonists LGD-1550 (Ligand) Immuno- interferon dexosometherapy (Anosys) modulators oncophage (Antigenics) pentrix (AustralianCancer Technology) GMK (Progenics) ISF-154 (Tragen) adenocarcinomavaccine (Biomira) cancer vaccine (Intercell) CTP-37 (AVI BioPharma)norelin (Biostar) IRX-2 (Immuno-Rx) BLP-25 (Biomira) PEP-005 (PeplinBiotech) MGV (Progenics) synchrovax vaccines (CTL Immuno) β-alethine(Dovetail) melanoma vaccine (CTL Immuno) CLL therapy (Vasogen) p21 RASvaccine (GemVax) Hormonal and estrogens prednisone antihormonalconjugated estrogens methylprednisolone agents ethinyl estradiolprednisolone chlortrianisen aminoglutethimide idenestrol leuprolidehydroxyprogesterone caproate goserelin medroxyprogesterone leuporelintestosterone bicalutamide testosterone propionate; fluoxymesteroneflutamide methyltestosterone octreotide diethylstilbestrol nilutamidemegestrol mitotane tamoxifen P-04 (Novogen) toremofine2-methoxyestradiol (EntreMed) dexamethasone arzoxifene (Eli Lilly)Photodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda)agents Theralux (Theratechnologies) lutetium texaphyrin (Pharmacyclics)motexafin gadolinium (Pharmacyclics) hypericin Tyrosine Kinase imatinib(Novartis) kahalide F (PharmaMar) Inhibitors leflunomide(Sugen/Pharmacia) CEP-701 (Cephalon) ZD1839 (AstraZeneca) CEP-751(Cephalon) erlotinib (Oncogene Science) MLN518 (Millenium) canertinib(Pfizer) PKC412 (Novartis) squalamine (Genaera) phenoxodiol ( ) SU5416(Pharmacia) trastuzumab (Genentech) SU6668 (Pharmacia) C225 (ImClone)ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210(Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447(Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth)IMC-1C11 (ImClone) EKB-569 (Wyeth)

Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin,netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems(e.g., imipenem/cislastatin), cephalosporins, colistin, methenamine,monobactams (e.g., aztreonam), penicillins (e.g., penicillin G,penicillin V, methicillin, natcillin, oxacillin, cloxacillin,dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin,piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, andvancomycin; and bacteriostatic agents such as chloramphenicol,clindanyan, macrolides (e.g., erythromycin, azithromycin,clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines(e.g., tetracycline, doxycycline, minocycline, demeclocyline), andtrimethoprim. Also included are metronidazole, fluoroquinolones, andritampin.

Enzyme inhibitors are substances which inhibit an enzymatic reaction.Examples of enzyme inhibitors include edrophonium chloride,N-methylphysostigmine, neostigmine bromide, physostigmine sulfate,tacrine, tacrine, 1-hydroxy maleate, iodotubercidin, p-bromotetramisole,10-(alpha-diethylaminopropionyl)-phenothiazine hydrochloride,calmidazolium chloride, hemicholinium-3,3,5-dinitrocatechol,diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II,3-phenylpropargylamine, N⁶-monomethyl-L-arginine acetate, carbidopa,3-hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl,hydroxylamine, iproniazid phosphate, 6-MeO-tetrahydro-9H-pyrido-indole,nialamide, pargyline, quinacrine, semicarbazide, tranylcypromine,N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride,3-isobutyl-1-methylxanthne, papaverine, indomethacind,2-cyclooctyl-2-hydroxyethylamine hydrochloride,2,3-dichloro-a-methylbenzylamine (DCMB),8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride,p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine,alpha-methyltyrosine, acetazolamide, dichlorphenamide,6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

Antihistamines include pyrilamine, chlorpheniramine, andtetrahydrazoline, among others.

Anti-inflammatory agents include corticosteroids, nonsteroidalanti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin,sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen,phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexatecoichicine, allopurinol, probenecid, and sulfinpyrazone.

Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprinehydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, andbiperiden.

Anti-spasmodics include atropine, scopolamine, oxyphenonium, andpapaverine.

Analgesics include aspirin, phenybutazone, idomethacin, sulindac,tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin,morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids(e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate,loperamide, morphine sulfate, noscapine, norcodeine, normorphine,thebaine, nor-binaltorphimine, buprenorphine, chlornaltrexamine,funaltrexaamione, nalbuphine, nalorphine, naloxone, naloxonazine,naltrexone, and naltrindole), procaine, lidocain, tetracaine anddibucaine.

Ophthalmic agents include sodium fluorescein, rose bengal, methacholine,adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase,betaxalol, pilocarpine, timolol, timolol salts, and combinationsthereof.

Prostaglandins are art recognized and are a class of naturally occurringchemically related, long-chain hydroxy fatty acids that have a varietyof biological effects.

Anti-depressants are substances capable of preventing or relievingdepression. Examples of anti-depressants include imipramine,amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine,doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

Growth factors are factors whose continued presence improves theviability or longevity of a cell. Trophic factors include, withoutlimitation, neutrophil-activating protein, monocyte chemoattractantprotein, macrophage-inflammatory protein, platelet factor, plateletbasic protein, and melanoma growth stimulating activity; epidermalgrowth factor, transforming growth factor (alpha), fibroblast growthfactor, platelet-derived endothelial cell growth factor, insulin-likegrowth factor (IGF, e.g., IGF-I or IGF-II), glial derived growthneurotrophic factor, ciliary neurotrophic factor, nerve growth factor,bone growth/cartilage-inducing factor (alpha and beta), bonemorphogenetic proteins (BMPs), interleukins (e.g., interleukininhibitors or interleukin receptors, including interleukin 1 throughinterleukin 10), interferons (e.g., interferon alpha, beta and gamma),hematopoietic factors, including erythropoietin, granulocyte colonystimulating factor, macrophage colony stimulating factor andgranulocyte-macrophage colony stimulating factor; tumor necrosisfactors, transforming growth factors (beta), including beta-1, beta-2,beta-3, transforming growth factors (alpha), inhibin, and activin; andbone morphogenetic proteins such as OP-1, BMP-2 and BMP-7.

Hormones include estrogens (e.g., estradiol, estrone, estriol,diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol,mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins(e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone,norgestrel), antiprogestin (mifepristone), androgens (e.g, testosteronecypionate, fluoxymesterone, danazol, testolactone), anti-androgens(e.g., cyproterone acetate, flutamide), thyroid hormones (e.g.,triiodothyronne, thyroxine, propylthiouracil, methimazole, andiodixode), and pituitary hormones (e.g., corticotropin, sumutotropin,oxytocin, and vasopressin). Hormones are commonly employed in hormonereplacement therapy and/or for purposes of birth control. Steroidhormones, such as prednisone, are also used as immunosuppressants andanti-inflammatories.

The biologically active agent is also desirably selected from the familyof proteins known as the transforming growth factors-beta (TGF-β)superfamily of proteins, which includes the activins, inhibins, and bonemorphogenetic proteins (BMPs). In an embodiment, the active agentincludes at least one protein selected from the subclass of proteinsknown generally as BMPs, which have been disclosed to have osteogenicactivity, and other growth and differentiation type activities. TheseBMPs include BMP proteins BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7,disclosed for instance in U.S. Pat. Nos. 5,108,922; 5,013,649;5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCTpublication WO91/18098; and BMP-9, disclosed in PCT publicationWO93/00432, BMP-10, disclosed in PCT application WO94/26893; BMP-11,disclosed in PCT application WO94/26892, or BMP-12 or BMP-13, disclosedin PCT application WO 95/16035; BMP-14; BMP-15, disclosed in U.S. Pat.No. 5,635,372; or BMP-16, disclosed in U.S. Pat. No. 5,965,403. OtherTGF-β proteins which may be useful as the active agent in the calciumphosphate compositions of the invention include Vgr-2, Jones et al.,Mol. Endocrinol. 6:1961 (1992), and any of the growth anddifferentiation factors (GDFs), including those described in PCTapplications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681;WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also usefulin the invention may be BIP, disclosed in WO94/01557; HP00269, disclosedin JP Publication number: 7-250688; and MP52, disclosed in PCTapplication WO93/16099. The disclosures of all of the above applicationsare incorporated herein by reference. A subset of BMPs which can be usedin the invention include BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10,BMP-12, BMP-13, BMP-14, and MP52. The active agent is most preferablyBMP-2, the sequence of which is disclosed in U.S. Pat. No. 5,013,649,the disclosure of which is incorporated herein by reference. Otherosteogenic agents known in the art can also be used, such asteriparatide (Forteo™), Chrysalin®, prostaglandin E2, LIM protein,osteogenin, or demineralized bone matrix (DBM), among others.

The biologically active agent may be synthesized chemically,recombinantly produced, or purified from a source in which thebiologically active agent is naturally found. The active agent, if aTGF-β, such as a BMP or other dimeric protein, may be homodimeric, ormay be heterodimeric with other BMPs (e.g., a heterodimer composed ofone monomer each of BMP-2 and BMP-6) or with other members of the TGF-βsuperfamily, such as activins, inhibins and TGF-β1 (e.g., a heterodimercomposed of one monomer each of a BMP and a related member of the TGF-βsuperfamily). Examples of such heterodimeric proteins are described forexample in Published PCT Patent Application WO 93/09229, thespecification of which is incorporated herein by reference.

Additional biologically active agents include the Hedgehog, Frazzled,Chordin, Noggin, Cerberus, and Follistatin proteins. These families ofproteins are generally described in Sasai et al., Cell 79:779-790 (1994)(Chordin); PCT Patent Publication WO94/05800 (Noggin); and Fukui et al.,Devel. Biol. 159:131 (1993) (Follistatin). Hedgehog proteins aredescribed in WO96/16668; WO96/17924; and WO95/18856. The Frazzled familyof proteins is a recently discovered family of proteins with highhomology to the extracellular binding domain of the receptor proteinfamily known as Frizzled. The Frizzled family of genes and proteins isdescribed in Wang et al., J. Biol. Chem. 271:4468-4476 (1996). Theactive agent may also include other soluble receptors, such as thetruncated soluble receptors disclosed in PCT patent publicationWO95/07982. From the teaching of WO95/07982, one skilled in the art willrecognize that truncated soluble receptors can be prepared for numerousother receptor proteins. The above publications are incorporated byreference herein.

The amount of the biologically active protein, e.g., an osteogenicprotein, that is effective to stimulate a desired activity, e.g.,increased osteogenic activity of present or infiltrating progenitor orother cells, will depend upon the size and nature of the defect beingtreated, as well as the carrier being employed. Generally, the amount ofprotein to be delivered is in a range of from about 0.1 to about 100 mg;preferably about 1 to about 100 mg; most preferably about 10 to about 80mg.

Standard protocols and regimens for delivery of the above-listed agentsare known in the art. Biologically active agents are introduced into theimplant material in amounts that allow delivery of an appropriate dosageof the agent to the implant site. In most cases, dosages are determinedusing guidelines known to practitioners and applicable to the particularagent in question. The exemplary amount of biologically active agent tobe included in the implant material of the invention is likely to dependon such variables as the type and extent of the condition, the overallhealth status of the particular patient, the formulation of the activeagent, and the bioresorbability of the delivery vehicle used. Standardclinical trials may be used to optimize the dose and dosing frequencyfor any particular biologically active agent.

In an embodiment of all aspects of the invention, the composition canadditionally contain autologous bone marrow or autologous plateletextracts.

In another embodiment of all of the above aspects, the PDGF and/or othergrowth factors can be obtained from natural sources, (e.g., platelets),or more preferably, produced by recombinant DNA technology. Whenobtained from natural sources, the PDGF and/or other growth factors canbe obtained from a biological fluid. A biological fluid includes anytreated or untreated fluid (including a suspension) associated withliving organisms, particularly blood, including whole blood, warm orcold blood, and stored or fresh blood; treated blood, such as blooddiluted with at least one physiological solution, including but notlimited to saline, nutrient, and/or anticoagulant solutions; bloodcomponents, such as platelet concentrate (PC), apheresed platelets,platelet-rich plasma (PRP), platelet-poor plasma (PPP), platelet-freeplasma, plasma, serum, fresh frozen plasma (FFP), components obtainedfrom plasma, packed red cells (PRC), buffy coat (BC); blood productsderived from blood or a blood component or derived from bone marrow; redcells separated from plasma and resuspended in physiological fluid; andplatelets separated from plasma and resuspended in physiological fluid.The biological fluid may have been treated to remove some of theleukocytes before being processed according to the invention. As usedherein, blood product or biological fluid refers to the componentsdescribed above, and to similar blood products or biological fluidsobtained by other means and with similar properties. In an embodiment,the PDGF is obtained from platelet-rich plasma (PRP). The preparation ofPRP is described in, e.g., U.S. Pat. Nos. 6,649,072, 6,641,552,6,613,566, 6,592,507, 6,558,307, 6,398,972, and 5,599,558, which areincorporated herein by reference.

In an embodiment of all aspects of the invention, the implant materialdelivers PDGF at the implant site for a duration of time greater than atleast 1 day. In several embodiments, the implant material delivers PDGFat the implant site for at least 7, 14, 21, or 28 days. Preferably, theimplant material delivers PDGF at the implant site for a time betweenabout 1 day and 7, 14, 21, or 28 days. In another embodiment, theimplant material delivers PDGF at the implant site for a time greaterthan about 1 day, but less than about 14 days.

By “bioresorbable” is meant the ability of the implant material to beresorbed or remodeled in vivo. The resorption process involvesdegradation and elimination of the original implant material through theaction of body fluids, enzymes or cells. The resorbed materials may beused by the host in the formation of new tissue, or it may be otherwisere-utilized by the host, or it may be excreted.

By “differentiation factor” is meant a polypeptide, including a chain ofat least 6 amino acids, which stimulates differentiation of one or moretarget cells into cells with cartilage or bone forming potential.

By “nanometer-sized particle” is meant a submicron-sized particle,generally defined as a particle below 1000 nanometers. A nanometer-sizedparticle is a solid particle material that is in an intermediate statebetween molecular and macron substances. A nanometer is defined as onebillionth of a meter (1 nanometer=10⁹ m). Nanometer material is known asthe powder, fiber, film, or block having nanoscale size.

By “periodontium” is meant the tissues that surround and support theteeth. The periodontium supports, protects, and provides nourishment tothe teeth. The periodontium consists of bone, cementum, alveolar processof the maxillae and mandible, periodontal ligament, and gingiva.Cementum is a thin, calcified layer of tissue that completely covers thedentin of the tooth root. Cementum is formed during the development ofthe root and throughout the life of the tooth and functions as an areaof attachment for the periodontal ligament fibers. The alveolar processis the bony portion of the maxilla and mandible where the teeth areembedded and in which the tooth roots are supported. The alveolar socketis the cavity within the alveolar process in which the root of the toothis held by the periodontal ligament. The bone that divides one socketfrom another is called the interdental septum. When multirooted teethare present, the bone is called the interradicular septum. The alveolarprocess includes the cortical plate, alveolar crest, trabecular bone,and the alveolar bone proper.

By “promoting growth” is meant the healing of bone, periodontium,ligament, or cartilage, and regeneration of such tissues and structures.Preferably, the bone, periodontium, ligament, or cartilage is damaged orwounded and requires regeneration or healing.

By “promoting periodontium growth” is meant regeneration or healing ofthe supporting tissues of a tooth including alveolar bone, cementum, andinterposed periodontal ligament, which have been damaged by disease ortrauma.

By “purified” is meant a growth or differentiation factor, e.g., PDGF,which, prior to mixing with a carrier substance, is 95% or greater byweight, i.e., the factor is substantially free of other proteins,lipids, and carbohydrates with which it is naturally associated. Theterm “substantially purified” refers to a lesser purity of factor,having, for example, only 5%-95% by weight of the factor, preferably65-95%. A purified protein preparation will generally yield a singlemajor band on a polyacrylamide gel. Most preferably, the purified factorused in implant materials of the invention is pure as judged byamino-terminal amino acid sequence analysis. The term “partiallypurified” refers to PDGF that is provided in the context of PRP, PPP,FFP, or any other blood product that requires collection and separation,e.g., by centrifugation, to produce.

By way of example, a solution having ˜1.0 mg/mL of PDGF, when ˜50% pure,constitutes ˜2.0 mg/mL of total protein.

The implant materials of this invention aid in regeneration ofperiodontium, at least in part, by promoting the growth of connectivetissue, bone, and cementum. The implant materials can be prepared sothat they directly promote the growth and differentiation of cells thatproduce connective tissue, bone, and cementum. Alternatively, theimplant materials can be prepared so that they act indirectly by, e.g.,attracting cells that are necessary for promoting the growth ofconnective tissue, bone, and cementum. Regeneration using a compositionof this invention is a more effective treatment of periodontal diseasesor bone wounds than that achieved using systemic antibiotics or surgicaldebridement alone.

The PDGF, polypeptide growth factors, and differentiation factors may beobtained from human tissues or cells, e.g., platelets, by solid phasepeptide synthesis, or by recombinant DNA technology. Thus, by the term“polypeptide growth factor” or “differentiation factor,” we mean tissueor cell-derived, recombinant, or synthesized materials. If the factor isa dimer, e.g., PDGF, the recombinant factor can be a recombinantheterodimer, made by inserting into cultured prokaryotic or eukaryoticcells DNA sequences encoding both subunits of the factor, and thenallowing the translated subunits to be processed by the cells to form aheterodimer (e.g., PDGF-AB). Alternatively, DNA encoding just one of thesubunits (e.g., PDGF B-chain or A-chain) can be inserted into cells,which then are cultured to produce the homodimeric factor (e.g., PDGF-BBor PDGF-AA homodimers). PDGF for use in the methods of the inventionincludes PDGF homo- and heterodimers, for example, PDGF-AA, PDGF-BB,PDGF-AB, PDGF-CC, and PDGF-DD, and combinations and derivatives thereof.

The concentration of PDGF or other growth factors of the invention canbe determined by using, e.g., an enzyme-linked immunoassay, as describedin, e.g., U.S. Pat. Nos. 6,221,625, 5,747,273, and 5,290,708,incorporated herein by reference, or any other assay known in the artfor determining protein concentration. When provided herein, the molarconcentration of PDGF is determined based on the molecular weight ofPDGF dimer (e.g., PDGF-BB; MW=approximately 25 kDa).

The methods and implant materials of the invention can be used to healbony wounds of mammals, e.g., fractures, implant recipient sites, andsites of periodontal disease. The implant materials promote connectivetissue growth and repair and enhance bone formation compared to naturalhealing (i.e., no exogenous agents added) or healing supplemented byaddition of systemic antibiotics. Unlike natural healing, conventionalsurgical therapy, or antibiotics, the implant materials of the inventionprompt increased bone, connective tissue (e.g., cartilage and ligament),and cementum formation when applied to damaged or diseased tissues or toperiodontal disease affected sites. The restoration of these tissuesleads to an improved prognosis for the affected areas. The ability ofthese factors to stimulate new bone formation also makes it applicablefor treating bony defects caused by other types of infection or surgicalor accidental trauma.

Other features and advantages of the invention will be apparent from thefollowing description of the embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G are photomicrographs showing the effect on bone formation 8weeks following treatment. FIG. 1A is a photomicrograph showing theeffect of surgery alone on bone formation. FIG. 1B is a photomicrographshowing the effect of β-TCP alone on bone formation. FIG. 1C is aphotomicrograph showing the effect of β-TCP+0.3 mg/mL PDGF on boneformation. FIG. 1D is a photomicrograph showing the effect of β-TCP+1.0mg/mL PDGF on bone formation. FIG. 1E is a photomicrograph showing theeffect of demineralized freeze dried bone allograft (DFDBA) alone onbone formation. FIG. 1F is a photomicrograph showing the effect ofdemineralized freeze dried bone allograft (DFDBA)+0.3 mg/mL PDGF on boneformation. FIG. 1G is a photomicrograph showing the effect ofdemineralized freeze dried bone allograft (DFDBA)+1.0 mg/mL on boneformation.

FIGS. 2A-2C are photomicrographs showing the effect on bone formation 16weeks following treatment. FIG. 2A is a photomicrograph showing theeffect of β-TCP alone on bone formation. FIG. 2B is a photomicrographshowing the effect of β-TCP+0.3 mg/mL PDGF on bone formation. FIG. 2C isa photomicrograph showing the effect of β-TCP+1.0 mg/mL PDGF on boneformation.

DETAILED DESCRIPTION

We now describe several embodiments of the invention. Two examplesdemonstrating the use of PDGF as a bone and periodontum healing agentare presented below.

EXAMPLES Example I Preparation of PDGF

Osseous wounds, e.g., following periodontal disease or trauma, aretreated and periodontium, including bone, cementum, and connectivetissue, are regenerated, according to the invention by combiningpartially purified or purified PDGF with any of the pharmaceuticallyacceptable carrier substances described above. Purified PDGF can beobtained from a recombinant source or from human platelets. Commerciallyavailable recombinant PDGF can be obtained from R&D Systems Inc.(Minneapolis, Minn.), BD Biosciences (San Jose, Calif.), and Chemicon,International (Temecula, Calif.). Partially purified and purified PDGFcan also be prepared as follows:

Five hundred to 1000 units of washed human platelet pellets aresuspended in 1M NaCl (2 ml per platelet unit) and heated at 100° C. for15 minutes. The supernatant is then separated by centrifugation and theprecipitate extracted twice with the 1 m NaCl.

The extracts are combined and dialyzed against 0.08M NaCl/0.01M sodiumphosphate buffer (pH 7.4) and mixed overnight at 4° C. with CM-SephadexC-50 equilibrated with the buffer. The mixture is then poured into acolumn (5×100 cm), washed extensively with 0.08M NaCl/0.01M sodiumphosphate buffer (pH 7.4), and eluted with 1M NaCl while 10 ml fractionsare collected.

Active fractions are pooled and dialyzed against 0.3M NaCl/0.01M sodiumphosphate buffer (pH 7.4), centrifuged, and passed at 4° C. through a2.5×25 cm column of blue sepharose (Pharmacia) equilibrated with 0.3MNaCl/0.01M sodium phosphate buffer (pH 7.4). The column is then washedwith the buffer and partially purified PDGF eluted with a 1:1 solutionof 1M NaCl and ethylene glycol.

The partially purified PDGF fractions are diluted (1:1) with 1M NaCl,dialyzed against 1M acetic acid, and lyophilized. The lyophilizedsamples are dissolved in 0.8M NaCl/0.01M sodium phosphate buffer (pH7.4) and passed through a 1.2×40 cm column of CM-Sephadex C-50equilibrated with the buffer. PDGF is then eluted with a NaCl gradient(0.08 to 1M).

The active fractions are combined, dialyzed against 1M acetic acid,lyophilized, and dissolved in a small volume of 1M acetic acid. 0.5 mlportions are applied to a 1.2×100 cm column of Biogel P-150 (100 to 200mesh) equilibrated with 1M acetic acid. The PDGF is then eluted with 1Macetic acid while 2 mL fractions are collected.

Each active fraction containing 100 to 200 mg of protein is lyophilized,dissolved in 100 mL of 0.4% trifluoroacetic acid, and subjected toreverse phase high performance liquid chromatography on a phenylBondapak column (Waters). Elution with a linear acetonitrile gradient (0to 60%) yields pure PDGF.

PDGF Made By Recombinant DNA Technology can be Prepared as Follows:

Platelet-derived growth factor (PDGF) derived from human plateletscontains two polypeptide sequences (PDGF-B and PDGF-A polypeptides;Antoniades, H. N. and Hunkapiller, M., Science 220:963-965, 1983).PDGF-B is encoded by a gene localized on chromosome 7 (Betsholtz, C. etal., Nature 320:695-699), and PDGF-A is encoded by the sis oncogene(Doolittle, R. et al., Science 221:275-277, 1983) localized onchromosome 22 (Dalla-Favera, R., Science 218:686-688, 1982). The sisgene encodes the transforming protein of the Simian Sarcoma Virus (SSV)which is closely related to PDGF-2 polypeptide. The human cellular c-sisalso encodes the PDGF-A chain (Rao, C. D. et al., Proc. Natl. Acad. Sci.USA 83:2392-2396, 1986). Because the two polypeptide chains of PDGF arecoded by two different genes localized in separate chromosomes, thepossibility exists that human PDGF consists of a disulfide-linkedheterodimer of PDGF-B and PDGF-A, or a mixture of the two homodimers(PDGF-BB homodimer and PDGF-AA homodimer), or a mixture of theheterodimer and the two homodimers.

Mammalian cells in culture infected with the Simian Sarcoma Virus, whichcontains the gene encoding the PDGF-A chain, were shown to synthesizethe PDGF-A polypeptide and to process it into a disulfide-linkedhomodimer (Robbins et al., Nature 305:605-608, 1983). In addition, thePDGF-A homodimer reacts with antisera raised against human PDGF.Furthermore, the functional properties of the secreted PDGF-A homodimerare similar to those of platelet-derived PDGF in that it stimulates DNAsynthesis in cultured fibroblasts, it induces phosphorylation at thetyrosine residue of a 185 kD cell membrane protein, and it is capable ofcompeting with human (¹²⁵I)-PDGF for binding to specific cell surfacePDGF receptors (Owen, A. et al., Science 225:54-56, 1984). Similarproperties were shown for the sis/PDGF-A gene product derived fromcultured normal human cells (for example, human arterial endothelialcells), or from human malignant cells expressing the sis/PDGF-2 gene(Antoniades, H. et al., Cancer Cells 3:145-151, 1985).

The recombinant PDGF-B homodimer is obtained by the introduction of cDNAclones of c-sis/PDGF-B gene into mouse cells using an expression vector.The c-sis/PDGF-B clone used for the expression was obtained from normalhuman cultured endothelial cells (Collins, T., et al., Nature216:748-750, 1985).

Use of PDGF

PDGF alone or in combination with other growth factors is useful forpromoting bone healing, bone growth and regeneration or healing of thesupporting structures of teeth injured by trauma or disease. It is alsouseful for promoting healing of a site of extraction of a tooth, formandibular ridge augmentation, or at tooth implant sites. Bone healingwould also be enhanced at sites of bone fracture or in infected areas,e.g., osteomyelitis, or at tumor sites. PDGF is also useful forpromoting growth and healing of a ligament, e.g., the periodontalligament, and of cementum.

In use, the PDGF or other growth or differentiation factor is applieddirectly to the area needing healing or regeneration. Generally, it isapplied in a resorbable or non-resorbable carrier as a liquid or solid,and the site then covered with a bandage or nearby tissue. An amountsufficient to promote bone growth is generally between 500 ng and 5 mgfor a 1 cm² area, but the upper limit is really 1 mg for a 1 cm² area,with a preferred amount of PDGF applied being 0.3 mg/mL.

Example II Periodontal Regeneration with rhPDGF-BB TreatedOsteoconductive Scaffolds

The effectiveness of PDGF in promoting periodontium and bone growth isdemonstrated by the following study.

In Vivo Dog Study

The beagle dog is the most widely used animal model for testing putativeperiodontal regeneration materials and procedures (Wikesjo et al., J.Clin. Periodontol. 15:73-78, 1988; Wikesjo et al., J. Clin. Periodontol.16:116-119, 1999; Cho et al., J. Periodontol. 66:522-530, 1995;Giannobile et al., J. Periodontol. 69:129-137, 1998; and Clergeau etal., J. Periodontol. 67:140-149, 1996). Plaque and calculus accumulationcan induce gingival inflammation that may lead to marginal bone loss andthe etiology of periodontitis in dogs and humans can be compared. Innaturally occurring disease, however, there is a lack of uniformitybetween defects. Additionally, as more attention has been given to oralhealth in canine breeder colonies, it has become impractical to obtainanimals with natural periodontal disease. Therefore, thesurgically-induced horizontal Class III furcation model has become oneof the most commonly used models to investigate periodontal healing andregeneration.

Beagle dogs with horizontal Class III furcation defects were treatedusing PDGF compositions of the invention, Fifteen adult beagle dogscontributed 60 treated defects. Forty-two defects were biopsied twomonths after treatment and fifteen defects were biopsied four monthsafter treatment

Defect Preparation

The “critical-size” periodontal defect model as described by numerousinvestigators was utilized (see, e.g., Wikesjo, 1988 and 1999, supra;Giannobile, supra, Cho, supra, and Park et al., J. Periodontol.66:462-477, 1995). Both mandibular quadrants in 16 male beagle dogs (2-3years old) without general and oral health problems were used. One monthprior to dosing, the animals were sedated with a subcutaneous injectionof atropine (0.02 mg/kg) and acepromazine (0.2 mg/kg) approximately 30minutes prior to being anesthetized with an IV injection ofpentobarbital sodium (25 mg/kg). Following local infiltration of thesurgical area with Lidocaine HCl plus epinephrine 1:100,000, fullthickness mucoperiosteal flaps were reflected and the first and thirdpremolars (P1 and P3) were extracted. Additionally, the mesial portionof the crown of the 1st molar was resected.

Alveolar bone was then removed around the entire circumference of P2 andP4, including the furcation areas using chisels and water-cooled carbideand diamond burs. Horizontal bone defects were created such that therewas a distance of 5 mm from the formix of the furcafion to the crest ofthe bone. The defects were approximately 1 cm wide, depending on thewidth of the tooth. The roots of all experimental teeth were planed withcurettes and ultrasonic instruments and instrumented with a tapereddiamond bur to remove cementum. After the standardized bone defects werecreated the gingival flaps were sutured to achieve primary closure. Theanimals were fed a soft diet and received daily chlorhexidine rinses forthe duration of the study.

Application of Graft Material

The periodontal defects of P2 and P4 in each mandibular quadrant of the15 animals were randomized prior to treatment using sealed envelopes.About four weeks after defect preparation, animals were re-anesthetizedas described above and full thickness flaps were reflected in bothmandibular quadrants. A notch was placed in the tooth root surfaces atthe residual osseous crest using a ½ round bur to serve as a futurehistologic reference point. The sites were irrigated with sterile salineand the roots were treated with citric acid as described previously forthe purpose of decontamination and removal of the smear layer (See,e.g., Cho, supra, and Park, supra). During this period an amount ofβ-TCP or DFDBA sufficient to fill the periodontal defect was saturatedwith a solution of rhPDGF-BB solution (0.3 or 1.0 mg/ml) and therhPDGF-BB/graft mixture was allowed to sit on the sterile surgical standfor about ten minutes. The rhPDGF-BB saturated graft was then packedinto the defect with gentle pressure to the ideal level of osseousregeneration.

After implantation of the graft material, the mucoperiosteal flaps weresutured approximately level to the cementoenamel junction (CEJ) usinginterproximal, interrupted 4.0 expanded polytetrafluoroethylene (ePTFE)sutures. Following suturing of the flaps chlorhexidine gluconate gel wasgently placed around the teeth and gingivae.

Treatment and Control Groups

Defects Received Either:

1. β-TCP

2. β-TCP plus rhPDGF-BB (0.3 mg/ml rhPDGF-BB)

3. β-TCP plus rhPDGF-BB (1.0 mg/ml rhPDGF-BB)

4. Dog DFDBA

5. Dog DFDBA plus rhPDGF-BB (0.3 mg/ml rhPDGF-BB)

6. Dog DFDBA plus rhPDGF-BB (1.0 mg/ml rhPDGF-BB)

7. Sham surgery (treated by open flap debridement only, no graft)

Six defects per treatment group were biopsied at two months (42 totalsites). In addition, five defects in treatment groups 1, 2, and 3 werebiopsied at four months (15 total sites).

TABLE 2 Experimental design NO. OF GROUP TEST NO. SITES TREATMENT TIMEPOINTS 1 11 β-TCP alone 8 & 16 weeks n = 6 for 8 wk n = 5 for 16 wk 2 11β-TCP + 0.3 mg/ml 8 & 16 weeks rhPDGF-BB n = 6 for 8 wk n = 5 for 16 wk3 11 β-TCP + 1.0 mg/ml 8 & 16 weeks rhPDGF-BB n = 6 for 8 wk n = 5 for16 wk 4 6 DFDBA alone 8 weeks 5 6 DFDBA + 0.3 8 weeks mg/ml rhPDGF-BB 66 DFDBA + 1.0 8 weeks mg/ml rhPDGF-BB 7 6 Surgery, no graft 8 weeks

Accordingly, at 8 weeks there are 7 groups divided among 42 sites in 11dogs. At 16 weeks, there are 3 groups divided among 15 sites in 4 dogs(one dog received two treatment surgeries staggered eight weeks apartand thus contributed two sites to each the 8 and 16 week time points).

Post-Surgical Treatment

The surgical sites were protected by feeding the dogs a soft diet duringthe first 4 weeks post-operative. To insure optimal healing, systemicantibiotic treatment with penicillin G benzathine was provided for thefirst two weeks and plaque control was maintained by daily irrigationwith 2% chlorhexidine gluconate throughout the experiment. Sutures wereremoved after 3 weeks.

Data Collection

Rationale for Data Collection Points

The eight week time point was chosen because this is the most commontime point reported for this model in the literature and therefore thereare substantial historical data. For example, Wikesjo et al., supra, andGiannobile et al., supra, also chose 8 weeks to assess the regenerativeeffects of BMP-2 and OP-1, respectively, in the same model.Additionally, Park et al., supra, evaluated the effect or rhPDGF-BBapplied directly to the conditioned root surface with and without GTRmembranes in the beagle dog model at 8 weeks. These studies, stronglysuggest that the 8 week period should be optimal for illustratingpotential significant effects among the various treatment modalities.

The sixteen week time point was chosen to assess long-term effects ofgrowth factor treatment. Previous studies (Park et al., supra) suggestthat by this time there is substantial spontaneous healing of theosseous defects. Nevertheless, it is possible to assess whetherrhPDGF-BB treatment leads to any unusual or abnormal tissue response,such as altered bone remodeling, tumorgenesis or root resorption.

Biopsies and Treatment Assessments

At the time of biopsy, the animals were perfused with 4%paraformaldehyde and sacrificed. The mandibles were then removed andplaced in fixative. Periapical radiographs were taken and the treatedsites were cut into individual blocks using a diamond saw. The coded(blinded) blocks were wrapped in gauze, immersed in a solution of 4%formaldehyde, processed, and analyzed.

During processing the biopsies were dehydrated in ethanol andinfiltrated and embedded in methylmethacrylate. Undecalcified sectionsof approximately 300 μm in thickness were obtained using a low speeddiamond saw with coolant. The sections were glued onto opalescentacrylic glass, ground to a final thickness of approximately 80 μm, andstained with toludine blue and basic fuchsin. Step serial sections wereobtained in a mesiodistal plane.

Histomorphometric analyses were performed on the masked slides. Thefollowing parameters were assessed:

1. Length of Complete New Attachment Apparatus (CNAA): Periodontalregeneration measured as the distance between the coronal level of theold bone and the coronal level of the new bone, including only that newbone adjacent to new cementum with functionally oriented periodontalligament between the new bone and new cementum.

2. New Bone Fill (NB): Measured as the cross-sectional area of new boneformed within the furcation.

3. Connective Tissue fill (CT): Measured as the area within thefurcation occupied by gingival connective tissue.

4. Void (VO): The area of recession where there is an absence of tissue.

Results

A. Clinical Observations

Clinically, all sites healed well. There was an impression that thesites treated with rhPDGF-BB healed more quickly, as indicated by thepresence of firm, pink gingivae within one week post-operatively. Therewere no adverse events experienced in any treatment group as assessed byvisual inspection of the treated sites. There appeared to be increasedgingival recession in groups that received β-TCP or DFDBA alone.

B. Radiographic Observations

Radiographically, there was evidence of increased bone formation at twomonths as judged by increased radiopacity in Groups 2, 3(β-TCP+rhPDGF-BB 0.3 and 1.0 mg/ml, respectively) and 6 (DFDBA+rhPDGF-BB1.0 mg/ml) compared to the other groups (FIGS. 1A-G). At four months,there was evidence of increased bone formation in all groups compared tothe two month time point. There was no radiographic evidence of anyabnormal bone remodeling, root resorption, or ankylosis in any group.

TABLE 3 Radiographic results. Rank order. QUALITATIVE ASSESSMENT OF BONEFILL AT 8 WKS* TREATMENT 6 β-TCP alone 1 β-TCP + 0.3 mg/ml rhPDGF 2β-TCP + 1.0 mg/ml rhPDGF 7 DFDBA alone 5 DFDBA + 0.3 mg/ml rhPDGF 3DFDBA + 1.0 mg/ml rhPDGF 4 Surgery, no graft *1 = most fill; 7 = leastfill

C. Histomorphometric Analyses:

Histomorphometric assessment of the length of new cementum, new bone,and new periodontal ligament (CNAA) as well as new bone fill, connectivetissue fill, and void space were evaluated and are expressed aspercentages. In the case of CNAA, values for each test group representthe CNAA measurements (length in mm)/total available CNAA length (inmm)×100%. Bone fill, connective tissue fill and void space wereevaluated and are expressed as percentages of the total furcation defectarea.

One-way analysis of variance (ANOVA) was used to test for overalldifferences among treatment groups, and pairwise comparisons were madeusing the student's t-test. Significant differences between groups werefound upon analyses of the coded slides. Table 4 shows the results attwo months.

TABLE 4 Two month histometric analyses % CNAA GROUP PERIODONTAL % %CONNECTIVE NO. TREATMENT REGENERATION BONE FILL TISSUE FILL % VOID 1β-TCP alone 37.0 ± 22.8 28.0 ± 29.5 36.0 ± 21.5 12.0 ± 17.9 ** 2 β-TCP +0.3 mg/ml 59.0 ± 19.1 84.0 ± 35.8 0.0 ± 0.0  8.0 ± 17.9 rhPDGF *, † †, ‡3 β-TCP + 1.0 mg/ml 46.0 ± 12.3 74.2 ± 31.7 0.0 ± 0.0 0.0 ± 0.0 rhPDGF *‡ 4 DFDBA alone 13.4 ± 12.0 6.0 ± 8.9 26.0 ± 19.5 30.0 ± 27.4 5 DFDBA +0.3 mg/ml 21.5 ± 13.3 20.0 ± 18.7 36.0 ± 13.4 18.0 ± 21.7 rhPDGF 6DFDBA + 1.0 mg/ml 29.9 ± 12.4 46.0 ± 23.0 26.0 ± 5.48  8.0 ± 13.04rhPDGF ≠ 7 Sham Surgery, 27.4 ± 15.0 34.0 ± 27.0  48.0 ± 35.64 10.0 ±22.4 no graft * Groups 2 and 3 significantly greater (p < 0.05) thanGroups 4 and 7. ** Group 1 significantly greater (p < 0.05) than Group4. † Group 2 significantly greater (p < 0.05) than Group 5. ‡ Groups 2and 3 significantly greater than Groups 1, 4 and 7. ≠ Group 6significantly greater than Group 4.

The mean percent periodontal regeneration (CNAA) in the surgery withoutgrafts and surgery plus β-TCP alone groups were 27% and 37%,respectively. In contrast, β-TCP groups containing rhPDGF-BB exhibitedsignificantly greater periodontal regeneration (p<0.05) than surgerywithout grafts or DFDBA alone (59% and 46% respectively for the 0.3 and1.0 mg/ml concentrations versus 27% for surgery alone and 13% for DFDBAalone). Finally, the β-TCP group containing 0.3 mg/ml rhPDGF-BBdemonstrated significantly greater periodontal regeneration (p<0.05)than the same concentration of rhPDGF-BB combined with allograft (59%versus 21%).

Bone fill was significantly greater (p<0.05) in the β-TCP+0.3 mg/mlrhPDGF-BB (84.0%) and the β-TCP+1.0 mg/ml rhPDGF-BB (74.2%) groups thanin the β-TCP alone (28.0%), surgery alone (34%) or DFDBA alone (6%)treatment groups. There was also significantly greater bone fill(p<0.05) for the β-TCP+0.3 mg/ml rhPDGF-BB group compared to theDFDBA+0.3 mg/ml rhPDGF-BB group (84% and 20% respectively).

The group of analyses examining the 8-week data from the DFDBA groupsand the surgery alone group (Groups 4, 5, 6, and 7) demonstrated nostatistically significant differences between the DFDBA groups andsurgery alone for periodontal regeneration (CNAA). There was a trendtoward greater regeneration for those sites treated with the 1.0 mg/mlrhPDGF-BB enhanced DFDBA versus DFDBA alone. There was significantlygreater bone fill (p<0.05) for sites treated with DFDBA+1.0 mg/mlrhPDGF-BB than DFDBA alone (46 and 6% respectively). There was a trendtoward greater bone fill for sites treated with DFDBA containing 0.3mg/ml rhPDGF-BB compared to DFDBA alone or surgery alone. However, sitestreated with DFDBA alone demonstrated less bone fill into the defectthan surgery alone (6 and 34%, respectively), with most of the defectbeing devoid of any fill or fill consisting of gingival (soft)connective tissue.

At four months following treatment, there remained significantdifferences in periodontal regeneration. β-TCP alone, as a result ofextensive ankylosis, resulted in 36% regeneration, while the sitestreated with β-TCP containing rhPDGF-BB had a mean regeneration of 58%and 49% in the 0.3 and 1.0 mg/ml rhPDGF-BB concentrations. Substantialbone fill was present in all three treatment groups. β-TCP aloneresulted in 70% bone fill, β-TCP plus 0.3 mg/ml rhPDGF yielded 100% fillwhile the 1.0 mg/ml rhPDGF group had 75% fill.

D. Histologic Evaluation

Histologic evaluation was performed for all biopsies except one, inwhich evaluation was not possible due to difficulties encountered duringprocessing.

Representative photomicrographs are shown in FIGS. 1A-G and 2A-C. FIG.1A shows results from a site treated with surgery alone (no grafts).This specimen demonstrates limited periodontal regeneration (new bone(NB), new cementum (NC), and periodontal ligament (PDL)) as evidenced inthe area of the notches and extending only a short distance coronally.The area of the furcation is occupied primarily by dense soft connectivetissue (CT) with minimal new bone (NB) formation.

For sites treated with β-TCP alone (FIG. 1B) there is periodontalregeneration, similar to that observed for the surgery alone specimen,that extends from the base of the notches for a short distancecoronally. As was seen in the surgery alone specimens, there was verylittle new bone formation with the greatest area of the furcation beingoccupied by soft connective tissue.

In contrast, FIG. 1C illustrates results obtained for sites treated withβ-TCP+0.3 mg/ml rhPDGF-BB. Significant periodontal regeneration is shownwith new bone, new cementum, and periodontal ligament extending alongthe entire surface of the furcation. Additionally, the area of thefurcation is filled with new bone that extends the entire height of thefurcation to the fornix.

Representative results for sites treated with β-TCP+1.0 mg/ml rhPDGF-BBare shown in FIG. 1D. While there is significant periodontalregeneration in the furcation, it does not extend along the entiresurface of the furcation. There is new bone formation present along withsoft connective tissue that is observed at the coronal portion of thedefect along with a small space which is void of any tissue (VO) at thefornix of the furcation.

FIGS. 2A, 2B, and 2C illustrate results obtained for the allografttreatment groups. Representative results for the DFDBA alone group (FIG.2A) shows very poor periodontal regeneration that is limited to the areaof the notches extending only slightly in a coronal direction. New boneformation is limited and consists of small amounts of bone formationalong the surface of residual DFDBA graft material (dark red stainingalong lighter pink islands). Additionally, the new bone is surrounded byextensive soft connective tissue that extends coronally to fill asignificant area within the furcation. Finally, a large void spaceextends from the coronal extent of the soft connective tissue to theformix of the furcation.

Histologic results for the DFDBA+0.3 and 1.0 mg/ml rhPDGF-BB are shownin FIGS. 2B and 2C, respectively. Both groups demonstrate greaterperiodontal regeneration compared to DFDBA alone with a complete newattachment apparatus (new bone, new cementum, and periodontal ligament)extending from the base of the notches in the roots for a short distancecoronally (arrows). They also had greater bone fill within the area ofthe furcation, although there was significant fill of the furcation withsoft connective tissue.

Conclusions

Based on the results of the study, treatment of a periodontal defectusing rhPDGF-BB at either 0.3 mg/mL or 1.0 mg/mL in combination with asuitable carrier material (e.g., β-TCP) results in greater periodontalregeneration than the current products or procedures, such as graftswith β-TCP or bone allograft alone, or periodontal surgery withoutgrafts.

Treatment with the 0.3 mg/mL and 1.0 mg/mL concentration of rhPDGFresulted in periodontal regeneration. The 0.3 mg/ml concentration ofrhPDGF demonstrated greater periodontal regeneration and percent bonefill as compared to the 1.0 mg/ml concentration of rhPDGF when mixedwith β-TCP.

β-TCP was more effective than allograft when mixed with rhPDGF-BB at anyconcentration. The new bone matured (remodeled) normally over time (0,8, and 16 weeks) in all groups. There was no increase in ankylosis orroot resorption in the rhPDGF groups. In fact, sites receiving rhPDGF-BBtended to have less ankylosis than control sites. This finding mayresult from the fact that rhPDGF-BB is mitogenic and chemotactic forperiodontal ligament cells.

Materials and Methods

Materials Utilized: Test and Control Articles

The β-TCP utilized had a particle-size (0.25 mm-1.0 mm) that wasoptimized for periodontal use. Based on studies using a canine model,administered β-TCP is ˜80% resorbed within three months and is replacedby autologous bone during the healing process.

The DFDBA was supplied by Musculoskeletal Transplant Foundation (MTF).The material was dog allograft, made by from the bones of a dog that waskilled following completion of another study that tested a surgicalprocedure that was deemed to have no effect on skeletal tissues.

Recombinant hPDGF-BB was supplied by BioMimetic Pharmaceuticals and wasmanufactured by Chiron, Inc, the only supplier of FDA-approved rhPDGF-BBfor human use. This rhPDGF-BB was approved by the FDA as a wound healingproduct under the trade name of Regranex®.

One ml syringes containing 0.5 ml of sterile rhPDGF-BB at two separateconcentrations prepared in conformance with FDA standards for humanmaterials and according to current applicable Good ManufacturingProcesses (cGMP). Concentrations tested included 0.3 mg/ml and 1.0mg/ml.

β-TCP was provided in vials containing 0.5 cc of sterile particles.

DFDBA was provided in 2.0 ml syringes containing 1.0 cc of sterile,demineralized freeze-dried dog bone allograft.

Material Preparation

At the time of the surgical procedure, the final implanted grafts wereprepared by mixing the rhPDGF-BB solution with the matrix materials.Briefly, an amount of TCP or allograft sufficient to completely fill theosseous defect was placed into a sterile dish. The rhPDGF-BB solutionsufficient to completely saturate the matrix was then added, thematerials were mixed and allowed to sit on the surgical tray for about10 minutes at room temperature prior to being placed in the osseousdefect.

A 10 minute incubation time with the β-TCP material is sufficient toobtain maximum adsorption of the growth factor (see Appendix A). This isalso an appropriate amount of time for surgeons in a clinical setting tohave prior to placement of the product into the periodontal defect.Similarly, in a commercial market, the rhPDGF-BB and the matrix materialcan be supplied in separate containers in a kit and that the materialscan be mixed directly before placement. This kit concept would greatlysimplify product shelf life/stability considerations.

Example III Use of PDGF for the Treatment of Periodontal Bone Defects inHumans

Recombinant human PDGF-BB (rhPDGF-BB) was tested for its effect on theregeneration of periodontal bone in human subjects. Two test groups wereadministered rhPDGF-BB at either 0.3 mg/mL (Group I) or 1.0 mg/mL (GroupII). rhPDGF-BB was prepared in sodium acetate buffer and administered ina vehicle of beta-tricalcium phosphate (β-TCP). The control group, GroupIII, was administered β-TCP in sodium acetate buffer only.

The objective of clinical study was to evaluate the safety andeffectiveness of graft material comprising β-TCP and rhPDGF-BB at either0.3 mg/mL or 1.0 mg/mL in the management of one (1) to three (3) wallintra-osseous periodontal defects and to assess its regenerativecapability in bone and soft tissue.

Study Design and Duration of Treatment

The study was a double-blind, controlled, prospective, randomized,parallel designed, multi-center clinical trial in subjects who requiredsurgical intervention to treat a bone defect adjacent to the naturaldentition. The subjects were randomized in equal proportions to resultin three (3) treatment groups of approximately 60 subjects each (180total). The duration of the study was six (6) months followingimplantation of the study device. The study enrolled 180 subjects.

Diagnosis and Main Entry Criteria

Male and female subjects, 25-75 years of age, with advanced periodontaldisease in at least one site requiring surgical treatment to correct abone defect were admitted to the study. Other inclusion criteriaincluded: 1) a probing pocket depth measuring 7 mm or greater at thebaseline visit; 2) after surgical debridement, 4 mm or greater verticalbone defect (BD) with at least 1 bony wall; 3) sufficient keratinizedtissue to allow complete tissue coverage of the defect; and, 4)radiographic base of defect at least 3 mm coronal to the apex of thetooth. Subjects who smoked up to 1 pack a day and who had teeth withClass I & II furcation involvement were specifically allowed.

Dose and Mode of Administration

All treatment kits contained 0.25 g of β-TCP (an active control) andeither 0.5 mL sodium acetate buffer solution alone (Group III), 0.3mg/mL rhPDGF-BB (Group I), or 1.0 mg/mL rhPDGF-BB (Group II).

Following thorough debridement and root planing, the test solution wasmixed with β-TCP in a sterile container, such that the β-TCP was fullysaturated. Root surfaces were conditioned using either tetracycline,EDTA, or citric acid. The hydrated graft was then packed into theosseous defect and the tissue flaps were secured with interdentalsutures to achieve complete coverage of the surgical site.

Effectiveness Measurement

The primary effectiveness measurement included the change in clinicalattachment level (CAL) between baseline and six months post-surgery(Group I vs. Group III). The secondary effectiveness measurementsconsisted of the following outcomes: 1) linear bone growth (LBG) and %bone fill (% BF) from baseline to six months post-surgery based on theradiographic assessments (Group I and Group II vs. Group III); 2) changein CAL between baseline and six months post-surgery (Group II vs. GroupIID; 3) probing pocket depth reduction (PDR) between baseline and sixmonths post-surgery (Group I and Group II vs. Group IID; 4) gingivalrecession (GR) between baseline and six months post-surgery (Group I andGroup II vs. Group III); 5) wound healing (WH) of the surgical siteduring the first three weeks post-surgery (Group I and Group II vs.Group IID; 6) area under the curve for the change in CAL betweenbaseline and three (3) and six (6) months (Group I and Group II vs.Group III); 7) the 95% lower confidence bound (LCB) for % BF at six (6)months post-surgery (Groups I, II, and III vs. demineralizedfreeze-dried bone allograft (DFDBA) as published in the literature;Parashis et al., J. Periodontol. 69:751-758, 1998); 8) the 95% LCB forlinear bone growth at six (6) months post-surgery (Groups I, II, and IIIvs. demineralized freeze-dried bone allograft (DFDBA) as published inthe literature; Persson et al., J. Clin. Periodontol. 27:104-108, 2000);9) the 95% LCB for the change in CAL between baseline and six (6) months(Groups I, II, and II vs. EMDOGAIN®-PMA P930021, 1996); and 10) the 95%LCB for the change in CAL between baseline and six (6) months (Groups I,II and III vs. PEPGEN P-15™-PMA P990033, 1999).

Statistical Methods

Safety and effectiveness data were examined and summarized bydescriptive statistics. Categorical measurements were displayed ascounts and percents, and continuous variables were displayed as means,medians, standard deviations and ranges. Statistical comparisons betweenthe test product treatment groups (Groups I and II) and the control(Group III) were made using Chi-Square and Fisher's Exact tests forcategorical variables and t-tests or Analysis of Variance Methods(ANOVA) for continuous variables. Comparisons between treatment groupsfor ordinal variables were made using Cochran-Mantel-Haenszel methods. Ap<0.05 (one sided) was considered to be statistically significant forCAL, LBG and % BF.

Safety data were assessed by the frequency and severity of adverseevents as evaluated clinically and radiographically. There were nosignificant differences between the three treatment groups at baseline.There were also no statistically significant differences observed in theincidence of adverse events (AEs; all causes) among the three treatmentgroups. The safety analysis did not identify any increased risk to thesubject due to implantation of the graft material.

Summary of Effectiveness Results

The results from the statistical analyses revealed both clinically andstatistically significant benefits for the two treatment groups (GroupsI and II), compared to the active control of β-TCP alone (Group III) andhistorical controls including DFDBA, EMDOGAIN®, and PEPGEN P-15™.

At three months post-surgery, a statistically significant CAL gain frombaseline was observed in favor of Group I versus Group II (p=0.041),indicating that there are significant early benefits of PDGF on the gainin CAL. At six months post-surgery, this trend continued to favor GroupI over Group III, although this difference was not statisticallysignificant (p=0.200). The area under the curve analysis (AUC) whichrepresents the cumulative effect (i.e. speed) for CAL gain betweenbaseline and six months approached statistical significance favoringGroup I in comparison to Group III (p=0.054). Further, the 95% lowerconfidence bound (LCB) analyses for all treatment groups substantiatedthe effectiveness of Groups I and II compared to the CAL gains observedat six (6) months for EMDOGAIN® and PEPGEN P-15™.

In addition to the observed clinical benefits of CAL, radiographicanalyses including Linear Bone Growth (LBG) and Percent Bone Fill (%BF), revealed statistically significant improvement in bone gain forGroups I and II vs. Group III. % BF was defined as the percent of theoriginal osseous defect filled with new bone as measuredradiographically. LBG showed significant improvement in Group I (2.5 mm)when compared to Group III (0.9 mm, p<0.001). LBG was also significantfor Group II (1.5 mm) when compared to Group III (p=0.021).

Percent Bone Fill (% BF) was significantly increased at six monthspost-surgical in Group I (56%) and Group II (34%) when compared to GroupII (18%), for a p<0.001 and p=0.019, respectively. The 95% lower boundof the confidence interval at six months post-surgery, for both linearbone growth and % bone fill, substantiated the effectiveness of Groups Iand II compared to the published radiographic results for DFDBA, themost widely used material for periodontal grafting procedures.

At three months, there was significantly less Gingival Recession (GR)(p=0.041) for Group I compared to Group III consistent with thebeneficial effect observed with CAL. No statistically significantdifferences were observed in PDR and GR at six months. Descriptiveanalysis of the number of sites exhibiting complete wound healing (WH)at three weeks revealed improvements in Group 1 (72%) vs. Group II (60%)and Group III (55%), indicating a trend toward improved healing.

To assess the cumulative beneficial effect for clinical and radiographicoutcomes, a composite effectiveness analysis was performed to determinethe percent of patients with a successful outcome as defined by CAL>2.7mm and LBG>1.1 mm at six (6) months. The CAL and LBG benchmarks ofsuccess were established by the mean levels achieved for theseparameters by the implanted grafts, as identified in the “EffectivenessMeasures” section above. The results showed that 61.7% of Group Ipatients and 37.9% of Group II patients met or exceeded the compositebenchmark for success compared to 30.4% of Group III patients, resultingin a statistically significant benefit of Group I vs. Group III(p<0.001). % BF revealed similar benefits for Group I (70.0%) vs. GroupIII (44.6%) for p-value of 0.003.

In summary, Group I achieved statistically beneficial results for CALand GR at three (3) months as well as LBG and % BF at six (6) months,compared to the β-TCP alone active control group (Group III). Theclinical significance of these results is further confirmed bycomparison to historical controls. It is concluded that PDGF-containinggraft material was shown to achieve clinical and radiographiceffectiveness by six months for the treatment of periodontal osseousdefects.

TABLE 5 Summary of PDGF Graft Effectiveness ENDPOINT GROUP I GROUP IIGROUP III CAL Gain (mm): 3 months 3.8 3.4 3.3 (p = 0.04)  (p = 0.40)CAL: AUC Analysis (mm × wk) 67.5 61.8 60.1 (p = 0.05)  (p = 0.35) CAL(mm): 95% LCB 6 months 3.3 3.2 3.1 (vs 2.7 mm for EMDOGAIN & 1.1 mm forPEPGEN) GR (mm): 3 months 0.5 0.7 0.9 (p = 0.04)  (p = 0.46) LBG (mm): 6months 2.5 1.5 0.9 (p < 0.001) (p = 0.02) % BF: 6 months 56.0 33.9 17.9(p < 0.001) (p = 0.02) Composite CAL-LBG 61.7% 37.9% 30.4% (p < 0.001)(p = 0.20) Analysis CAL-% BF 70.0% 55.2% 44.6% (% Success) (p = 0.003)(p = 0.13)

Graft material (i.e., β-TCP) containing PDGF at 0.3 mg/mL and at 1.0mg/mL was shown to be safe and effective in the restoration of alveolarbone and clinical attachment around teeth with moderate to advancedperiodontitis in a large, randomized clinical trial involving 180subjects studied for up to 6 months. These conclusions are based uponvalidated radiographic and clinical measurements as summarized below.

Consistent with the biocompatibility data of the PDGF-containing graftmaterial, discussed above, and the historical safe use of eachindividual component (i.e., β-TCP alone or PDGF alone), the studyrevealed no evidence of either local or systemic adverse effects. Therewere no adverse outcomes attributable to the graft material, which wasfound to be safe.

Conclusion

Implantation of β-TCP containing PDGF at either 0.3 mg/mL or 1.0 mg/mLwas found to be an effective treatment for the restoration of softtissue attachment level and bone as shown by significantly improved CALat 3 months compared to the active control. Our findings are alsoconsistent with the AUC analysis that showed an improvement in CAL gainbetween baseline and six months. Implantation of β-TCP containing PDGFat either 0.3 mg/mL or 1.0 mg/mL was also found to be an effectivetreatment based on significantly improved LBG and % BF compared to theactive control. Significantly improved clinical outcomes as shown by thecomposite analysis of both soft and hard tissue measurements compared tothe β-TCP alone active control also demonstrate the effectiveness of thetreatment protocol described above. Finally, the results ofadministering β-TCP containing PDGF at either 0.3 mg/mL or 1.0 mg/mLwere found to exceed established benchmarks of effectiveness bothclinically and radiographically.

The results of this trial together with extensive and confirmatory datafrom in vitro, animal and human studies demonstrate that PDGF-containinggraft material stimulates soft and hard tissue regeneration inperiodontal defects, although the effects were more significant whenPDGF in the range of 0.1 to 1.0 mg/mL (e.g., 0.1 mg/mL, 0.3 mg/mL, or1.0 mg/mL) was administered in the graft material. Moreover, PDGFadministered in the graft material in the amount of 0.3 mg/mLeffectively regenerated soft tissue and bone.

Other embodiments are within the following claims.

What is claimed is:
 1. A method for promoting growth of bone, ligament,or cartilage of a mammal comprising administering to the mammal animplant material consisting of: (i) platelet-derived growth factor(PDGF) at a concentration in a range of about 0.1 mg/mL to about 1.0mg/mL in a solution, and (ii) a pharmaceutically acceptable solidcarrier, wherein the pharmaceutically acceptable solid carrier is (a)calcium phosphate or (b) calcium phosphate and collagen; wherein saidcarrier is capable of absorbing an amount of the PDGF solution that isequal to at least about 25% of its own weight; wherein the calciumphosphate is bioresorbable, comprises interconnected pores, and consistsof particles with a particle size range of about 100 to about 5000 μm;and wherein the implant material promotes the growth of the bone,ligament, or cartilage.
 2. The method of claim 1, wherein the PDGF has aconcentration of about 0.3 mg/mL.
 3. The method of claim 1, wherein thePDGF has a concentration of about 1.0 mg/mL.
 4. The method of claim 1,wherein the PDGF is in a pharmaceutically acceptable liquid carrier. 5.The method of claim 1, wherein the PDGF is PDGF-AA, PDGF-BB, PDGF-CC, orPDGF-DD, or a combination thereof or a derivative thereof.
 6. The methodof claim 5, wherein the PDGF is recombinant human (rh) PDGF-BB.
 7. Themethod of claim 1, wherein the calcium phosphate is tricalciumphosphate, hydroxyapatite, poorly crystalline hydroxyapatite, amorphouscalcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate,heptacalcium phosphate, calcium pyrophosphate dihydrate, calciumpyrophosphate, or octacalcium phosphate.
 8. The method of claim 7,wherein the calcium phosphate is tricalcium phosphate and the tricalciumphosphate is β-tricalcium phosphate (β-TCP).
 9. The method of claim 8,wherein the β-TCP is in a particle form and the particles have a size inthe range of about 100 to about 3000 μm.
 10. The method of claim 9,wherein the β-TCP particles have a size in the range of about 250 toabout 2000 μm.
 11. The method of claim 1, wherein the implant materialdelivers the PDGF to the bone, ligament, or cartilage for at least 1 dayfollowing administration.
 12. The method of claim 1, wherein the PDGF isreleased from the implant material upon administration at an averagerate of less than or equal to 300 μg/day.
 13. A method for promotinggrowth of bone, ligament, or cartilage of a mammal comprisingadministering to the mammal an implant material consisting of: (i) PDGFat a concentration in a range of about 0.1 mg/mL to about 1.0 mg/mL, and(ii) a pharmaceutically acceptable solid carrier, wherein thepharmaceutically acceptable solid carrier is (a) calcium phosphate or(b) calcium phosphate and collagen; wherein said carrier has a porosityof greater than 40%; wherein the calcium phosphate is bioresorbable,comprises interconnected pores, and consists of particles with aparticle size range of about 100 to about 5000 μm; and wherein theimplant material promotes the growth of the bone, ligament, orcartilage.
 14. The method of claim 13, wherein the concentration of PDGFis about 0.3 mg/ml or about 1.0 mg/ml.
 15. The method of claim 13,wherein the PDGF is in a pharmaceutically acceptable liquid carrier. 16.The method of claim 13, wherein the PDGF is PDGF-AA, PDGF-BB, PDGF-CC,or PDGF-DD, or a combination thereof or a derivative thereof.
 17. Themethod of claim 16, wherein the PDGF is recombinant human (rh) PDGF-BB.18. The method of claim 13, wherein the calcium phosphate is tricalciumphosphate, hydroxyapatite, poorly crystalline hydroxyapatite, amorphouscalcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate,heptacalcium phosphate, calcium pyrophosphate dihydrate, calciumpyrophosphate, or octacalcium phosphate.
 19. The method of claim 18,wherein the calcium phosphate is tricalcium phosphate and the tricalciumphosphate is β-tricalcium phosphate (β-TCP).
 20. The method of claim 19,wherein the β-TCP consists of particles having a size in the range ofabout 250 to about 2000 μm.
 21. The method of claim 13, wherein theimplant material delivers the PDGF to the bone, ligament, or cartilagefor at least 1 day following administration.
 22. The method of claim 13,wherein the PDGF is released from the implant material uponadministration at an average rate of less than or equal to 300 μg/day.23. A method for promoting growth of bone, ligament, or cartilage of amammal comprising the steps of: (1) surgically exposing the bone,ligament, or cartilage treatment site; (2) removing organic matter fromthe target tissue at the treatment site; (3) implanting an implantmaterial at the treatment site; wherein the implant material consistsof: (i) PDGF at a concentration in a range of about 0.1 mg/mL to about1.0 mg/mL in a solution and a pharmaceutically acceptable solid carrier,wherein the pharmaceutically acceptable solid carrier is (a) calciumphosphate or (b) calcium phosphate and collagen; wherein said carrier iscapable of absorbing an amount of the PDGF solution that is equal to atleast about 25% of its own weight; and wherein the calcium phosphate isbioresorbable, comprises interconnected pores, and consists of particleswith a particle size range of about 100 to about 5000 μm; or (ii) PDGFat a concentration in a range of about 0.1 mg/mL to about 1.0 mg/mL in asolution and a pharmaceutically acceptable solid carrier, wherein thepharmaceutically acceptable solid carrier is (a) calcium phosphate or(b) calcium phosphate and collagen; wherein said carrier has a porosityof greater than 40%; and wherein the calcium phosphate comprisesinterconnected pores, and consists of particles with a particle size ofabout 100 to about 5000 μm; and (4) covering the implant site with abandage or nearby tissue.
 24. The method of claim 23, wherein the stepof removing organic matter from the target tissue at the treatment sitecomprises debridement of the target tissue.
 25. The method of claim 23,wherein the implant material is prepared by: placing thepharmaceutically acceptable solid carrier in a sterile dish; adding thePDGF solution sufficient to saturate the solid carrier; mixing the solidcarrier and the PDGF solution to create an implant material; andallowing the implant material to sit for a sufficient amount of time toallow the solid carrier to adsorb or absorb the PDGF.
 26. The method ofclaim 25, wherein the step of allowing the implant material to sit for asufficient amount of time to allow the solid carrier to adsorb or absorbthe PDGF consists of allowing the implant material to sit for about 10minutes at room temperature prior to being implanted.
 27. The method ofclaim 23, wherein the PDGF solution contains PDGF at a concentration ofabout 0.3 mg/mL.
 28. The method of claim 23, wherein PDGF is applied tothe treatment site in an amount equal to between 500 ng and 5 mg per 1cm² of treatment area.
 29. The method of claim 23, wherein PDGF isapplied to the treatment site in an amount equal to between 500 ng and 1mg per 1 cm² of treatment area.
 30. The method of claim 23, wherein thePDGF is obtained from natural sources or is recombinant.
 31. The methodof claim 23, wherein the PDGF is rhPDGF-BB.
 32. The method of claim 23,wherein the implant material is packed into a defect site at thetreatment site.
 33. The method of claim 32, wherein the implant materialis packed into an osseous defect site in an amount sufficient tocompletely fill the osseous defect.
 34. The method of claim 23, whereinthe treatment site is irrigated with sterile saline prior toimplantation of the implant material.
 35. The method of claim 23,wherein the method further includes administering an antibiotictreatment following the procedure.
 36. The method of claim 23, whereinthe method involves periodontal treatment and comprises the steps of:(1) creating a surgical flap to expose the treatment site; (2) treatinga tooth root with citric acid; (3) implanting the implant material; (4)replacing the flap; and (5) applying chlorhexidine gluconate gel gentlyaround the tooth and gingivae following replacement of the flap.
 37. Themethod of claim 23, wherein said method is done as part of a toothextraction, ridge augmentation, esthetic grafting, sinus lift, or bonefracture treatment, or as part of the treatment of an implant recipientsite, periodontal disease site, or bony defect.